title: Astrocyte cultures exhibit P2X7 receptor channel opening in the absence of exogenous ligands.
abstract: P2X7 receptors (P2X7Rs) gate the opening of large channels when activated by ATP or other ligands. P2X7Rs are expressed by astrocytes in culture and by reactive astrocytes in vivo, and astrocytes in culture have been shown to release glutamate and ATP in response to P2X7R activation. However, P2X7Rs are activated by ATP only at concentrations greater than 1 mM. The conditions under which astrocyte P2X7Rs would be activated in vivo are, thus, unclear. Here we show that astrocytes in culture exhibit basal P2X7R activity. Primary mouse astrocytes were found to take up the P2X7R permeant dyes YO-PRO-1 (YP) and propidium iodide in absence of any added ligands. By contrast, cultured rat astrocytes took up very little YP, consistent with their much lower level of P2X7R expression. The uptake by mouse astrocytes was inhibited by oxATP, suramin, KN-62 and brilliant blue G, and by siRNA knock-down of P2X7R. Astrocyte uptake of YP was also inhibited by phenol red at concentrations above 50 muM, suggesting that phenol red present in standard cell culture media may influence P2X7R channel activity. Treatment with apyrase, an enzyme that degrades extracellular ATP, partially decreased YP uptake in astrocytes. Conversely, exposure to the ectonucleotidase inhibitor ARL67156 enhanced YP uptake and astrocytes plated without contiguous neighboring astrocytes showed reduced basal YP uptake. These results suggest that the basal uptake of YP may be due to activation of P2X7R by release of ATP by astrocytes themselves into intercellular spaces.
authors.affiliation: Department of Neurology, University of California, San Francisco and Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA, USA. nagasawa@mb.kyoto-phu.ac.jp
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nagasawa
authors.forename: Carole
authors.initials: C
authors.lastname: Escartin
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Swanson
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001081:Apyrase / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism*; D001557:Benzenesulfonates / Q000494:pharmacology; D001583:Benzoxazoles / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D006801:Humans; D008024:Ligands; D051379:Mice; D010637:Phenolsulfonphthalein / Q000494:pharmacology; D011419:Propidium / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D011806:Quinolinium Compounds / Q000378:metabolism; D034741:RNA, Small Interfering; D051381:Rats; D017208:Rats, Wistar; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D013498:Suramin / Q000494:pharmacology
chemical_list: C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; D001557:Benzenesulfonates; D001583:Benzoxazoles; D008024:Ligands; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C546470:P2rx7 protein, rat; D058919:Purinergic P2 Receptor Antagonists; D011806:Quinolinium Compounds; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C089813:YO-PRO 1; D011419:Propidium; D013498:Suramin; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D001081:Apyrase; C006796:brilliant blue; D010637:Phenolsulfonphthalein